Mdm2 is required to negatively regulate p53 activity at the peri-implantation stage of early mouse development. However, the absolute requirement for Mdm2 throughout embryogenesis and in organogenesis is unknown. To explore Mdm2–p53 signaling in osteogenesis, Mdm2-conditional mice were bred with Col3.6-Cre–transgenic mice that express Cre recombinase in osteoblast lineage cells. Mdm2-conditional Col3.6-Cre mice die at birth and display multiple skeletal defects. Osteoblast progenitor cells deleted for Mdm2 have elevated p53 activity, reduced proliferation, reduced levels of the master osteoblast transcriptional regulator Runx2, and reduced differentiation. In contrast, p53-null osteoprogenitor cells have increased proliferation, increased expression of Runx2, increased osteoblast maturation, and increased tumorigenic potential, as mice specifically deleted for p53 in osteoblasts develop osteosarcomas. These results demonstrate that p53 plays a critical role in bone organogenesis and homeostasis by negatively regulating bone development and growth and by suppressing bone neoplasia and that Mdm2-mediated inhibition of p53 function is a prerequisite for Runx2 activation, osteoblast differentiation, and proper skeletal formation.

The Mdm2 gene is amplified in approximately onethird of human sarcomas and overexpressed in a variety of other human cancers. Mdm2 functions as an oncoprotein, in part, by acting as a negative regulator of the p53 tumor suppressor protein. Multiple spliced forms of Mdm2 transcripts have been observed in human tumors; however, the contribution of these variant transcripts to tumorigenesis is unknown. In this report, we isolate alternative splice forms of Mdm2 transcripts from sarcomas that spontaneously arise in Mdm2-overexpressing mice, including Mdm2-b, the splice form most commonly observed in human cancers. Transduction of Mdm2-b into a variety of cell types reveals that Mdm2-b promotes p53-independent cell growth, inhibits apoptosis, and up-regulates the RelA subunit of NFB. Furthermore, expression of Mdm2-b induces tumor formation in transgenic mice. These results identify a p53-independent role for Mdm2 and determine that an alternate spliced form of Mdm2 can contribute to formation of cancer via a p53-independent mechanism. These findings also provide a rationale for the poorer prognosis of those patients presenting with tumors harboring multiple Mdm2 transcripts.

Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.Although genetic and biochemical studies clearly indicate that Mdm2 and MdmX are key regulators of p53 activity, there are distinct differences in their mechanisms of p53 inhibition. Mdm2 forms a complex with p53 and functions as an E3 ligase to target p53 for ubiquitination and proteosomal degradation (16,18,25), thereby inhibiting p53's transactivation of genes whose products are involved in the regulation of cell growth and apoptosis (47). MdmX complexes with p53 and inhibits p53 transactivation without altering p53 stability (11,46), and in contrast to that of Mdm2, MdmX expression is not regulated by p53. Regardless of these differences, Mdm2 and MdmX act as critical negative regulators of p53 function in development. The developmental block imposed by the loss of Mdm2 or MdmX can be relieved by the deletion of p53 (9,21,28,30,32) or by Mdm2 amplification (20,22) or, in the case of MdmX, partially rescued by the deletion of the p53 downstream effector p21 (43). These data indicate that the primary role of Mdm2 or MdmX in development is to regulate p53.The amplification and overexpression of either Mdm2 (31) or MdmX (7, 36) have been observed in a variety of human cancers, including sarcoma, glioma, and, in the case of MdmX, retinoblastoma (24), suggesting that either Mdm2 or MdmX can function as an oncogene to inhibit p53 activity and promote tumorigenesis. Since many of these Mdm-overexpressing tumors retain wild-type p53 alleles, the reactivation of p53 by small-molecule inhibition of the Mdm2-p53 or MdmX-p53 interaction is an attractive strategy for treating these cancers (26,27).The results of experiments in vitro or in vivo involving the forced overexpression of Mdm proteins suggest that Mdm2 and MdmX may also have p53-independent roles in promoting cell growth (13,23,29,38,41). However, molecular targets for Mdm2 or MdmX activity other than p53 have yet to be confirmed. Furthermore, it remains unclear if physiologic levels of eit...

The Runt-related transcription factor, Runx2, is essential for osteogenesis and is controlled by both distal (P1) and proximal (P2) promoters. To understand Runx2 function requires determination of the spatiotemporal activity of P1 and P2 to Runx2 protein production. We generated a mouse model in which the P1-derived transcript was replaced with a lacZ reporter allele, resulting in loss of P1-derived protein while simultaneously allowing discrimination between the activities of the two promoters. Loss of P1-driven expression causes developmental defects with cleidocranial dysplasia-like syndromes that persist in the postnatal skeleton. P1 activity is robust in preosteogenic mesenchyme and at the onset of bone formation but decreases as bone matures. Homozygous Runx2-P1 lacZ/lacZ mice have a normal life span but exhibit severe osteopenia and compromised bone repair in adult mice because of osteoblastic defects and not increased osteoclastic resorption. Gene expression profiles of bone, immunohistochemical studies, and ex vivo differentiation using calvarial osteoblasts and marrow stromal cells identified mechanisms for the skeletal phenotype. The findings indicate that P1 promoter activity is necessary for generating a threshold level of Runx2 protein to commit sufficient osteoprogenitor numbers for normal bone formation. P1 promoter function is not compensated via the P2 promoter. However, the P2 transcript with compensatory mechanisms from bone morphogenetic protein (BMP) and Wnt signaling is adequate for mineralization of the bone tissue that does form. We conclude that selective utilization of the P1 and P2 promoters enables the precise spatiotemporal expression of Runx2 necessary for normal skeletogenesis and the maintenance of bone mass in the adult.Runx2 is the master regulator of both osteoblast and terminal chondrocyte differentiation and is essential for in vivo bone formation and mineralization (1, 2). Runx2 is strongly expressed in mesenchymal condensations of the developing skeleton (2) during endochondral bone formation (3). A large number of bone-related genes are regulated by Runx2 including Runx2 and its targets that contribute to the bone matrix: osteocalcin (OC), 6 osteopontin, bone sialoprotein, and alkaline phosphatase (AP). Runx2 also contributes to bone turnover through regulation of osteoprotegerin and receptor activator of nuclear factor -B ligand (RANKL) and maturation of the growth plate by expression of vascular endothelial growth factor (VEGF) and collagen type X (4 -10).Promoter switching is a common developmental mechanism used to control the gene expression levels and the functional activities of several genes in osteoblasts (e.g. collagen type I (Col1␣1) and parathyroid hormone-related protein) (11-13). The two distinct promoters of Runx2 may specifically regulate the dynamic process of bone development by controlling spatiotemporal expression of Runx2. The proximal P2 promoter (Runx2 P2) regulates the type I isoform (designated Runx2-I), which begins with the amino acids MRIPV an...

The p19ARF gene product responds to oncogenic stresses by interfering with the inhibitory effects of Mdm2 on p53, thus enhancing p53 activity and its antiproliferative functions. The absence of p19 ARF in the mouse leads to early tumor susceptibility, presumably in part due to decreased p53 activity. To examine the tumorigenic cooperativity of p19 ARF , Mdm2, and p53 in vivo, p19 ARF -deficient mice were crossed first to p53-deficient mice and then to Mdm2 transgenic mice. The progeny were monitored for tumors. Cooperativity between p19 ARF and p53 deficiencies in accelerating tumor formation was observed for most genotypes except p53À/À p19 ARF À/À mice. p53À/À p19 ARF À/À mice had a tumor incidence similar to p53À/À mice. In this context, tumor suppression by ARF appears to be primarily p53 dependent. The majority of the p19 ARF þ /À tumors deleted the wildtype p19 ARF allele, in agreement with the previous studies, suggesting that p19 ARF is a classic 'two hit' tumor suppressor. In a p53 þ /À background, however, all p19 ARF þ /À tumors retained a wildtype ARF allele and most also retained wildtype p53. In the second cross between p19 ARF -deficient and Mdm2 transgenic mice, cooperativity in tumor incidence between Mdm2 overexpression and ARF deficiency was observed, consistent with the role of p19 ARF in negatively regulating Mdm2 activity. These experiments further demonstrate in vivo the inter-relationships of the p19 ARF -Mdm2-p53 signaling axis in tumor suppression.

The Mdm2 and Mdm4 genes are amplified and overexpressed in a variety of human cancers and encode structurally related oncoproteins that bind to the p53 tumor suppressor protein and inhibit p53 activity. Mice deleted for either Mdm2 or Mdm4 die during embryogenesis, and the developmental lethality of either mouse model can be rescued by concomitant deletion of p53. However, the phenotypes of Mdm2 and Mdm4-deficient mice suggest that Mdm2 and Mdm4 play nonoverlapping roles in regulating p53 activity during development, with Mdm2 regulating p53-mediated cell death and Mdm4 regulating p53-mediated inhibition of cell growth. Here, we describe complete rescue of Mdm4-deficient mice by expression of an Mdm2 transgene, and demonstrate that Mdm2 can regulate both p53-mediated apoptosis and inhibition of cell growth in the absence of Mdm4 in primary cells. Furthermore, deletion of Mdm4 enhances the ability of Mdm2 to promote cell growth and tumor formation, indicating that Mdm4 has antioncogenic properties when Mdm2 is overexpressed

Mdm4 (MdmX) is a p53-binding protein that shares structural similarities with Mdm2 and has been proposed to be a negative regulator of p53 function. Like Mdm2, the absence of Mdm4 has recently been found to induce embryonic lethality in mice that is rescued by p53 deletion. Mdm4-null embryos are reduced in size and die at midgestation, and Mdm4-deficient embryos and embryonic fibroblasts displayed reduced rates of cell proliferation. The p53-induced, cyclin-dependent kinase inhibitor p21 is strongly upregulated in Mdm4-null embryos and cells. Here, we report that deletion of p21 delays the midgestation lethality observed in Mdm4-null mice, suggesting that Mdm4 downregulates p53-mediated suppression of cell growth. Surprisingly, the absence of p21 also uncovers an antiproliferative effect of Mdm4 on cell growth in vitro and in Mdm4-heterozygous mice. These results indicate that p21 is a downstream modifier of Mdm4, and provides genetic evidence that Mdm4 can function to regulate cell growth both positively and negatively.

The Inhibitor of Growth (ING) gene family encodes structurally related proteins that alter chromatin to regulate gene expression and cell growth. The initial member, ING1, has also been proposed to function as a tumor suppressor in human cancer based on its ability to suppress cell growth and transformation in vitro. Mouse Ing1 produces two proteins (p31 and p37) from differentially spliced transcripts. We have recently generated p37Ing1b -null mice and observed spontaneous follicular B-cell lymphomagenesis in this model to show that ING proteins can function in vivo as tumor suppressors. In this present report, we examine the role of p37Ing1b in the regulation of B-cell growth and explore the relationship between p37Ing1b and p53-mediated tumor suppression. Our results indicate that p37Ing1b inhibits the proliferation of B cells and follicular B cells regardless of p53 status, and loss of p53 greatly accelerates the rate of B-cell lymphomagenesis in p37Ing1b -null mice. However, in contrast to the highly penetrant follicular B-cell lymphomas observed in p37Ing1b -null mice, mice lacking both p37Ing1b and p53 typically present with aggressive diffuse large B-cell lymphomas (DLBL). Analysis of marker gene expression in p37Ing1b /p53 null tumors indicates that the double-null mice develop both nongerminal center and germinal center B-cell-like DLBL, and also documents up-regulation of nuclear factor-KB activity in p37Ing1b /p53-null B cells and B-cell tumors. These results confirm that p53 mutation is an important mechanistic step in the formation of diffuse large B-cell lymphomas and reveals a p53-independent role for Ing1b in suppressing B-cell tumorigenesis. [Cancer Res 2008;68(21):8705-14]